1. Islet Isolation
The insulin producing tissue of the pancreas, the islets of Langerhans, constitutes between about one and two percent of the mass of the pancreas. The isolation of the islets from the remainder of the pancreatic tissue is desirable for laboratory purposes and for transplantation purposes. Transplantation of islets is looked to as a possible treatment for diabetes. Transplanting islets rather than an intact pancreas or pancreatic segments offers several advantages, including the ease of transplantation, the possibility of in vitro treatment to prevent rejection without immunosuppression, the elimination of the pancreatic exocrine function (the secretion of digestive substances from the host tissue), the possibility of cryopreservation of the tissue for subsequent use, and the possibility of xenografts.
In an early method of islet separation, chopped pancreatic fragments are mixed with collagenase and incubated at 37xc2x0 C. (reviewed in Scharp, World Journal of Surgery 8:143-151 (1984)). The collagenase breaks down or digests the pancreatic tissue, freeing the islets. The collagenase also acts on the islets, so that the islets released early in the process are broken down into single cells. If the process is stopped to protect the islets released early, many islets remain trapped in pancreatic fragments. Therefore only a fraction of the available intact islets are released by this method. This process is particularly ineffective for the isolation of islets from the pancreata of large animals such as humans, dogs, or pigs.
Laboratory islet isolation from rodent pancreata was greatly improved by the discovery that mechanical distension of rodent pancreata increased islet yield by causing mechanical separation of islets from the pancreas tissue. After distension the pancreas is chopped for collagenase digestion. The beneficial effect of this same type of mechanical distension has also been noticed in large animals.
Horaguchi and Merrell, Diabetes 30:455-58 (1982) developed a method for perfusing the dog pancreas with collagenase via the pancreatic duct. Subsequently, a process involving ductal distension of the pancreas with a solution containing collagenase was developed (U.S. Pat. No. 4,868,121; incorporated herein by reference). Inflation or distension of the pancreas is believed to cause some mechanical rupturing of the exocrine tissue or partial separation of the islets from the exocrine tissue, making subsequent collagenase digestion easier.
2. Sonication
Sound waves have been used in the past to aggregate cells and to disrupt cells. For example, ultrasound has recently been used to aggregate cells as a purification procedure. Kilburn, D G, et al., xe2x80x9cEnhanced sedimentation of mammalian cells following acoustic aggregation,xe2x80x9d Biotechnol.Bioeng. 34:559-62 (1989). In this procedure, cells which are not sufficiently heavy to precipitate out of solution are caused to aggregate by exposure to ultrasound. The aggregates then precipitate out of the solution. This procedure uses a standing wave to aggregate the cells, and the procedure is performed in an echo chamber to create and maintain the standing wave.
Ultrasound has also long been used to disrupt cells. For example, exposure of cells to ultrasound is used to lyse the cells to isolate the nucleic acid contained inside. Crouse, C A, et al., xe2x80x9cExtraction of DNA from forensic-type sexual assault specimens using simple, rapid sonication proceduresxe2x80x9d BioTechniques 15:641-42,644-48 (1993). This procedure uses very concentrated sound waves to disrupt the cell structure. The ultrasonic field is applied at a localized spot, such as a microtip of an emitter.
The present invention is an improvement on the process for isolating cells, such as islets of Langerhans, which incorporates sonication of the organ, such as the pancreas, as a method for dissociating the cells from other non-desired tissue. The inventors have discovered that sonication of the pancreas in conjunction with collagenase treatment results in a high degree of dissociation of the islet cells that maintain a high degree of integrity. The invention can be applied to the isolation of specific cell types from many different types of organs.
The invention is an improved method for the isolation of specific viable cell types from surrounding organ tissue. The technique has specifically been applied to the isolation of islets of Langerhans cells from a pig pancreas as described below in the preferred embodiment. However, the invention is also applicable to the isolation of cell types from other organs and other animals (e.g., cells from organs from transgenic animals, islets from human pancreata). Other potential applications include the isolation of medullary cells from adrenal glands, and the isolation of hepatocytes from liver to be used, for example, as bioartificial liver systems. The organ is harvested and prepared as necessary, such as by removal of undesired segregated tissue or cells. The invention relies on the use of sound waves to accelerate tissue dissociation. The cells released from the dissociated tissue remain intact and viable, allowing separation of desired cells from unwanted tissue. Thus, this invention differs from the prior art wherein cells are either aggregated or disrupted. Implementation of the invention entails three steps.
Tissue dissociating agents will typically include tissue degrading enzymes such as collagenase, trypsin, neutral It protease or dispase, and other proteolytic enzymes, with the preferred embodiment demonstrating the use of collagenase. However, the tissue dissociating agents are not necessarily limited to enzymes. Other examples of tissue dissociating agents are chelating agents for the dissociation of fetal tissue. The length of time required for treatment with dissociating agents will vary depending on the type of the agent, the concentration of agent, and the temperature at which treatment is conducted. Treatment is allowed to proceed until a sufficient amount of tissue has dissociated without causing undue damage to released cells or cellular aggregates. Preferably at least 40%, more preferably at least 75%, and most preferably at least 90% of the tissue is dissociated, while less than 50%, more preferably less than 25%, and most preferably less than 10% of the cells are functionally damaged by treatment with the dissociating agents.
The preferred embodiment below describes the treatment of a pancreas via ductal distension, a method fully described in U.S. Pat. No. 4,868,121. That is a method in which the tissue dissociating effect of the treatment agent is enhanced by is injection of the agent into the pancreas to cause tension that results in some mechanical rupturing of the exocrine tissue or partial separation of the islets from the exocrine tissue. However, the invention is not limited to this form of treatment. Other possible types of the treatment would include chopping the organ smaller into pieces and incubation with a tissue dissociating agent, or use of a dissociating agent with mechanical agitation such as incubation of the organ with marbles in a shaking container. In the preferred embodiment described below, enzyme treatment and sonication occur simultaneously.
The sonication step as described in the preferred embodiment was accomplished with a sonicating waterbath. However, it should be appreciated that other types of sonication methods could also be used. These would include acoustic horns, piezo-electric crystals, or any other method of generating stable sound waves, such as with sonication probes. In the preferred embodiment described below, sonication was conducted at about 43 kHz for approximately 20 minutes. Under approximately these same conditions, a sonication frequency of between about 40 kHz to 50 kHz is likely to be equally effective. However, a fairly wide range of frequencies, from subaudio to ultrasound (between about 7 Hz to 40 MHz, preferably between 7 Hz and 20 MHz) would be expected to give sound-enhanced tissue dissociation. Additionally, variations in the type of sonication include pulsing versus continuous sonication.
The sound waves created by the sound source must be at sufficiently low power so as not to disrupt the cells being isolated. The sonication source is run at a power level of between 10xe2x88x924 and about 10 watts/cm2. See xe2x80x9cBiological Effects of Ultrasound: Mechanisms and Clinical Implications,xe2x80x9d National Council on Radiation Protection and Measurements (NCRP) Report No. 74, NCRP Scientific Committee No. 66: Wesley L. Nyborg, chairman; 1983; NCRP, Bethesda, Md. The sonicating water bath discussed above works at about 0.9 watt/cm2.
The tissue to be sonicated is present in a container which will hold the tissue and dissociated cells in a fluid and which is transparent to ultrasound waves. To avoid contamination of the tissue and cells, a closed container is preferred. Additionally, use of a light-transparent container will allow visual monitoring of the progress of dissociation. Further, use of a malleable container will allow tactile monitoring. In the preferred embodiment, a self-sealing polyethylene bag is used as the container for the organ that is placed in the sonicating water bath. Other types of enclosed malleable containers could also be used, or other containers such as a plastic beaker. The frequency and power of the sonication can be adjusted to accommodate significant changes in the type of container, the volume of buffer, or in the mass of material being sonicated.
Further, the conditions for sonication are such that a standing wave is not created. In the preferred embodiment described herein, the chamber of the sonicating water bath has rounded edges so as not to create a standing wave. Additionally, presence of the irregularly shaped, acoustically dense organ or tissue in the device impedes the production of a standing wave. Thus, according to the invention, the device for delivery of the sound waves, in combination with the tissue or organ to which the sound waves are being applied, are preferably configured to avoid the production of a standing wave.
It should be appreciated that the invention encompasses the use of these steps in other orders, such as partially overlapping of steps one and two, or tissue digestion prior to sonication.
Finally, once the cells of interest have become dissociated from the surrounding organ tissue by treatment with dissociating agents and sonication, they must be separated from the other organ tissue. There are a wide variety of techniques that can be used to accomplish this step. These include various techniques for mechanical disruption of the tissue such as aspiration through needles, maceration, and/or filtration. Such techniques for mechanical disruption are preferably accompanied by a concentration/purification step such as either centrifugation or use of a density gradient flush-out to separate the desired cells from the remaining tissue when the cells have a different density. Islets can be separated in this manner from other denser acinar tissue. The islet cells are then typically further purified using standard density gradient methods such as Percoll(copyright) (a colloidal PVP coated silica, available from Sigma) or Ficoll(copyright) (a non-ionic synthetic polymer of sucrose, available from Sigma) gradients. See Ballinger, W F and Lacy, P E, xe2x80x9cTransplantation of Intact Pancreatic Islets in Rats,xe2x80x9d Surgery 72:175-186 (1972), which is incorporated herein by reference.